The present invention relates to a process for enzymatic macromolecularization of phenolic compounds or aromatic amine compounds and to applications of the macromolecules obtained by the process.
More particularly, the present invention provides a process for producing phenolic compounds or aromatic amine compounds which have increased molecular weights by reacting phenolic compounds or aromatic amine compounds with an enzyme having a polyphenol oxidizing activity in the alkaline pH region, and applications of the reaction method of increasing the molecular weight of phenolic compounds or aromatic amine compounds utilizing the above-described catalytic activity of the enzyme in the alkali regions to obtain thickeners, stabilizers, coagulants, emulsifiers, dispersants, water retainers, antioxidants, adhesives, concrete admixtures, dyes, deodorants, smell eliminators, coating materials, petroleum recovering agents, soil conditioners, blow-applied seed bearing surface soil stabilizers, agricultural chemical spreaders, feeding stuff binders, bactericides, antimicrobial agents, viral infection inhibitors, bioadhesion preventives, biotic repellents, insecticides, poultices, ink bases and wood treating agents; a method of producing these various agents; a method of waste water disposal; a method of deoxygenation; and methods of treating wood, concrete and soil, respectively.
Hitherto, it has been known that phenolic compounds etc. can be macromolecularized by utilizing an enzyme such as laccase or polyphenol oxidase produced by Basidiomycotina or Deuteromycotina (Journal of Biotechnology, 13, 229-241, 1990 and etc. so on). However, the laccases or polyphenol oxidases produced by fungi have their optimal reaction pH in the acidic region so that the reaction must be carried out in pH region ranging from acidic to neutral in order to catalyze or accelerate the macromolecularization reaction utilizing these enzymes and in addition, the rate of the macromolecularization reaction is not high enough. Also, the natural organic compounds with which these enzymes react are mainly polyphenolic compounds and, hence, the reaction must be carried out in the pH region ranging from acidic to neutral because the optimal reaction pH of the enzyme is in the acidic region despite the fact that the polyphenolic compounds have solubilities which decrease in the pH region from acidic to neutral, resulting in a defect that it is impossible to efficiently macromolecularize polyphenolic compounds in high concentrations. Further, although many polyphenolic compounds are accelerated their autooxidation in the alkaline pH region, enzymatic oxidative macromolecularization has been carried out in the pH region ranging from acidic to neutral, resulting in a defect that the autooxidation cannot be utilized effectively.
Further, it has been known that phenolic compounds, etc. can be macromolecularized with bilirubin oxidase, too, and this reaction can be utilized in the macromolecularization of lignin and dying of cotton (WO95-01426, and JP-A-6-316874). However, in the prior art using bilirubin oxidase, the enzyme-catalytic macromolecularization of phenolic compounds, etc., is carried out in the pH region ranging from acidic to neutral and therefore the macromolecularization reaction rate is not sufficiently high or there is no description suggesting that the macromolecularization reaction of phenolic compounds is accelerated in the alkaline pH region by bilirubin oxidase.
An object of the present invention is to provide a process of enzyme-catalytically macromolecularizing phenolic compounds or aromatic amine compounds in the alkaline pH region.
Another object of the present invention is to provide a process of producing thickeners, stabilizers, coagulants, emulsifiers, dispersants, water retainers, antioxidants, adhesives, concrete admixtures, dyes, coating materials, petroleum recovering agents, soil conditioners, blow-applied seed bearing surface soil stabilizers, deodorants, smell eliminators, agricultural chemical spreaders, feeding stuff binders, bactericides, antimicrobial agents, viral infection inhibitors, bioadhesion preventives, biotic repellents, insecticides, poultices, ink bases and wood treating agents, comprising the step of efficiently macromolecularizing phenolic compounds or aromatic amine compounds using an enzyme having a polyphenol oxidizing activity in the alkaline pH region.
Still another object of the present invention is to provide thickeners, stabilizers, coagulants, emulsifiers, dispersants, water retainers, antioxidants, adhesives, concrete admixtures, dyes, coating materials, petroleum recovering agents, soil conditioners, blow-applied seed bearing surface soil stabilizers, deodorants, smell eliminators, agricultural chemical spreaders, feeding stuff binders, bactericides, antimicrobial agents, viral infection inhibitors, bioadhesion preventives, biotic repellents, insecticides, poultices, ink bases and wood treating agents, comprising the macromolecules obtained by efficiently macromolecularizing the above-described phenolic compounds or aromatic amine compounds.
Yet another object of the present invention is to provide a method of treating waste water containing phenolic compounds or aromatic amine compounds, in which the above-described compounds are disposed from the waste water by utilizing the above-described macromolecularization reaction according to the present invention.
Further, yet another object of the present invention is to provide a method of deoxygenation in which the above-described macromolecularization reaction according to the present invention is utilized to remove dissolved oxygen.
Further, object of the present invention is to provide a method of treating wood utilizing the above-described macromolecularization reaction according to the present invention.
Further, an object of the present invention is to provide a method of treating concrete utilizing the above-described macromolecularization reaction according to the present invention.
Furthermore, an object of the present invention is to provide a method of improving the soil utilizing the above-described macromolecularization reaction according to the present invention.
The present inventors have made intensive investigation in order to develop a process of efficiently macromolecularizing phenolic compounds or aromatic amine compounds. As a result, they have now found that, surprisingly, use of a suitable enzyme having a polyphenol oxidizing activity in the alkaline region, particularly in the alkaline pH region not lower than pH 8, results in the achievement of efficient macromolecularization of phenolic compounds or aromatic amine compounds, thus completing the present invention.
Accordingly, the present invention provides the followings:
1) A process of producing phenolic compounds or aromatic amine compounds having increased molecular weights, characterized by comprising allowing an enzyme having a polyphenol oxidizing activity to act on phenolic compounds or aromatic amine compounds in the alkaline pH region to macromolecularize them.
2) The process as described in 1) above, characterized in that the macromolecularization is carried out in the alkaline pH region of not lower than pH 8.
3) The process as described in 1) or 2) above, characterized in that as the enzyme having a polyphenol oxidizing activity is used one or more of catechol oxidase, laccase, polyphenol oxidase, ascorbic acid oxidase or bilirubin oxidase.
4) The process as described in any one of 1) to 3), wherein use is made of an enzyme having a polyphenol oxidizing activity obtained by cultivating a bacterium belonging to the genus Bacillus.
5) The process as described in 4) above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating Bacillus licheniformis or Bacillus natto. 
6) The process as described in 5) above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating Bacillus licheniformis SD3003 (Accession No. FERM BP-5801).
7) The process as described in any one of 1) to 3) above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating a fungus belonging to the genus Myrothecium.
8) The process as described in 7) above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating Myrothecium verrucaria or Myrothecium roridum. 
9) The process as described in 8) above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme obtained by cultivating Myrothecium verrucaria SD3001 (Accession No. FERM BP-5520) or Myrothecium roridum SD3002 (Accession No. FERM BP-5523).
10) The process as described in any one of 1) to 9) above, wherein the enzyme having a polyphenol oxidizing activity is an enzyme which has an optimal reaction pH in the alkaline region of not lower than pH 7.5 when the activity thereof is measured with syringaldazine.
11) The process as described in any one of 1) to 10) above, wherein the phenolic compound is lignin or a lignin derivative.
12) The process as described in 11) above, wherein the lignin derivative is lignosulfonic acid.
13) The process as described in any one of 1 to 10) above, wherein the phenolic compound is flavonoid.
14) The process as described in any one of 1) to 13) above, characterized in that the macromolecularization reaction is carried out by adding one or more of a quinone compound, unsaturated fatty acid, unsaturated alcohol or an unsaturated alkyl compound to the phenolic compound or aromatic amine compound.
15) The process as described in any one of 1) to 14) above, wherein an antimicrobial compound, an antiviral compound, a biotic repellent compound, an insecticidal compound or a metal ion coexists.
16) The process as described in any one of 1) to 15) above, wherein the macromolecularization is carried out at a temperature of 0 to 100xc2x0 C.
17) Thickeners, stabilizers, coagulants, emulsifiers, dispersants, water retainers, antioxidants, adhesives, concrete admixtures, dyes, coating materials, petroleum recovering agents, soil conditioners, blow-applied seed bearing surface soil stabilizers, deodorants, smell eliminators, agricultural chemical spreaders, feeding stuff binders, bactericides, antimicrobial agents, viral infection inhibitors, bioadhesion preventives, biotic repellents, insecticides, poultices, ink bases or wood treating agents, comprising macromolecular compound produced by the process as described in any one of 1) to 16) above.
18) A process of producing thickeners, stabilizers, coagulants, emulsifiers, dispersants, water retainers, antioxidants, adhesives, concrete admixtures, dyes, coating materials, petroleum recovering agents, soil conditioners, blow-applied seed bearing surface soil stabilizers, deodorants, smell eliminators, agricultural chemical spreaders, feeding stuff binders, bactericides, antimicrobial agents, viral infection inhibitors, bioadhesion preventives, biotic repellents, insecticides, poultices, ink bases or wood treating agents, comprising the step of macromolecularizing the phenolic compounds or aromatic amine compounds as described in any one of 1) to 16) above.
19) A method of disposing of waste water, characterized by comprising macromolecularizing phenolic compounds or aromatic amine compounds in waste water in accordance with the method as described in any one of 1) to 16) above and removing it from the waste water.
20) A deoxygenating agent for use in the alkaline pH region, characterized by comprising a phenolic compound or aromatic amine compound and the enzyme having a polyphenol oxidizing activity as described in any one of 1) to 16) above.
21) A method of treating wood, characterized by comprising impregnating wood with an enzyme having a polyphenol oxidizing activity together with a phenolic compound or aromatic amine compound and macromolecularizing the phenolic compound or aromatic amine compound in the wood.
22) A method of treating concrete, characterized by comprising adding to concrete an enzyme having a polyphenol oxidizing activity together with a phenolic compound or aromatic amine compound and macromolecularizing the phenolic compound or aromatic amine compound in the concrete.
23) A method of treating soil, characterized by comprising adding to soil an enzyme having a polyphenol oxidizing activity together with a phenolic compound or aromatic amine compound and macromolecularizing the phenolic compound or aromatic amine compound in the soil.
[Polyphenol Oxidase]
The enzymes used for the purposes of the present invention may be any enzyme that has a polyphenol oxidizing activity at alkaline pH values. Examples of such an enzyme include polyphenol oxidases such as catechol oxidase, laccase, polyphenol oxidase, ascorbic acid oxidase, bilirubin oxidase, or the like produced by microorganisms, for example fungi or bacteria, or plants. Besides these enzymes, any enzyme protein that has a polyphenol oxidizing activity at alkaline pH values may also be used in the present invention.
As the enzyme to be used in the present invention, having a polyphenol oxidizing activity in the alkaline pH region, those enzymes are desirable which have optimal reaction pH for polyphenol oxidation reaction in the alkaline region of not lower than pH 7.5 in order to carry out efficient macromolecularization reaction. More specifically, it is desirable that such enzymes have optimal reaction pH in the alkaline region of not lower than pH 7.5 when the activity thereof is measured with syringaldazine that is described later.
Examples of the microorganisms which produce the enzymes used for the purposes of the present invention include the followings.
As fungi, there can be cited those strains which belong to the genera falling in Deuteromycotina, i.e., Aspergillus, Botrytis, Myrothecium, Penicillium, Pestalotia, Rhizoctonia, Tricoderma, preferably Aspergillus nidulans, Botrytis cinerea, Myrothecium roridum, Myrothecium verrucaria, Myrothecium prestonii, Myrothecium leucotrichum, Penicillium sclerotiorum, Penicillium janthinellum, Pestalotia palmarum, Rhizoctonia praticola, Tricoderma resii, and Tricoderma viride. Of these, particularly preferred are Myrothecium verrucaria SD3001 (Deposited under Accession No. FERM P-14955 at Research Institute of Biotechnological and Industrial Science, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan on May 29, 1995 and transferred to international deposition under Accession No. FERM BP-5520 on Apr. 24, 1996) or Myrothecium roridum SD3002 (Deposited under Accession No. FERM P-15255 at Research Institute of Biotechnological and Industrial Science, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan on Oct. 29, 1995 and transferred to international deposition under Accession No. FERM BP-5523 on Apr. 24, 1996).
Other preferred fungi include those strains which belong to the genera falling in Basidiomycotina, i.e., Pleurotus, Lentinus, Schizophyllum, Armillariella, Flammulina, Agaricus, Coprinus, Phanerochaete, Phlebia, Lenzites, Melanoleuca, Pholiota, Stereumu, Polyporus, Polyporellus, Microporus, Fomitopsis, Pycnoporus, Trametes, Coriolus, Daedaleopsis, Rigidoporus, Fomes, Ganoderma, Trachyderma, Hymenochaete, and Inonotus, preferably Pleurotus cornucopiae, Pleurotus osteratus, Lentinus edodes, Schizophyllum commune, Armillariella mellea, Flammulina velutipes, Agaricus bisporus, Coprinus comatus, Coprinus cinereus, Coprinus congregatus, Phanerochaete chrysosporium, Phlebia radiata, Lenzites betulina, Melanoleuca verrucipes, Pholiota nameko, Stereumu hirsutum, Polyporus squamosus, Polyporellus badius, Microporus flabelliformis, Fomitopsis pinicola, Pycnoporus coccineus, Trametes orientalis, Coriolus versicolor, Coriolus hirsutus, Daedaleopsis tricolor, Rigidoporus zonalis, Fomes fomentarius, Ganoderma lucidum, Trachyderma tsunodae, Hymenochaete rubiginosa, and Inonotus mikadoi. 
Other preferred fungi include those strains which belong to the genera falling in Ascomycotina, i.e., Podospora, Neurospora, and Monocillium, preferably Podospora anserina, Neurospora crassa, and Monocillium indicum. 
Preferred bacteria include those strains belonging to Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus firmus, Bacillus licheniformis, Bacillus natto, Bacillus pumilus, Bacillus sphaericus, and Bacillus subtilis, preferably Bacillus licheniformis, and Bacillus natto. Of these, particularly preferred is Bacillus licheniformis SD3003 (Deposited under Accession No. FERM P-15383 at Research Institute of Biotechnological and Industrial Science, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan on Dec. 28, 1995 and transferred to international deposition under Accession No. FERM BP-5801 on Jan. 28, 1997).
Other preferred bacteria include those strains which belong to the genera Azospirillum, preferably Azospirillum lipoferum or those strains which belong to the genera falling in Actinomycetales, for example, Streptomyces, preferably Streptomyces antibioticus, or Aerobacter, preferably Aerobacter aerogenes. 
Some preferred plants which contains enzymes used in the present invention include Acer pseudoplatanum, Dioscorea, Abelmoschus, Psidium, Helianthus, potato, apple, pumpkin, cucumber, wheat, alfalfa, etc.
[Preparation of Enzymes]
The enzymes used in the present invention can be obtained by cultivating strains belonging to the above-described microorganisms, for example fungi or bacteria, and variants thereof. Besides, they can also be prepared by utilizing genetically engineered microorganisms. That is, the enzymes can be produced by cultivating, under conditions enabling expression of enzyme proteins, host cells transformed with an expression vector which includes a DNA vector having a replication initiator codon for replicating a vector in the host organism, the vector having inserted therein a DNA sequence encoding the above-described enzyme protein together with suitable promoter, operator, and terminator DNA sequences having an enzyme expressing function in the host organism, or host cells transformed by incorporating in the host cell DNA, a DNA sequence encoding the above-described enzyme together with suitable promoter, operator, and terminator DNA sequences which have an enzyme expressing function in the host organism, followed by recovering the enzyme protein from the culture medium.
The DNA fragments encoding the enzyme protein according to the present invention can be obtained by conventional methods such as the method in which cDNA or genome library from a strain belonging to the above-described microorganisms, for example fungi or bacteria, is used as an isolation source, and a target DNA fragment is identified using as a probe which is an oligonucleotide synthesized based on the amino acid sequence of the enzyme protein according to the present invention, the method in which a clone expressing the activity of an oxidase is screened, or the method in which a clone is screened which produces a protein that reacts with an antibody against the above-described enzyme protein.
It is possible to prepare the enzyme protein according to the present invention by extraction from seeds, fruits, leaves or the like of the above-described plants.
Further, in the cultivation of the strains belonging to fungi or bacteria and variants thereof for obtaining the enzyme protein according to the present invention, there can be used synthetic medium and nutrition medium containing organic carbon sources and organic nitrogen sources which are employed conventionally. In the case of cultivation, it is desirable that Cu2+ ions be added in amounts of 0.001 mM to 10 mM, preferably 0.01 mM to 1 mM, as metal salt.
When it is secreted outside the cells of fungi or bacteria, the polyphenol oxidase according to the present invention can be recovered from the culture medium by a well-known method. The recovery procedure includes a series of operations such as removal of cells from the culture medium by centrifugation, filtration or membrane separation, and chromatography, for example ion exchange chromatography. Also, membrane concentration with ultrafiltration membrane is effectively employed. When it is accumulated inside the cells of fungi or bacteria or when it exists inside plant tissues, the enzyme protein can be recovered from the microbial cells or plant tissues by a well-known method. The recovery procedure includes a series of operations such as mechanical rupture of the tissue by homogenization, isolation and extraction of an enzyme protein solution by centrifugation, filtration or membrane separation, and chromatography, for example ion exchange chromatography. Also, membrane concentration with ultrafiltration membrane is effectively employed.
[Measurement Method of Activity]
In the present invention, the measurement of polyphenol oxidizing activity of the enzyme protein having a polyphenol oxidizing activity was carried out by conducting the reaction in an aqueous solution containing 20 ppm of syringaldazine and 100 mM Tris-HCl buffer or potassium phosphate buffer at an optimal reaction pH at 20xc2x0 C. and measuring the absorbance at 525 nm. The amount of activity in which 1 xcexcmol/minute of syringaldazine is oxidized was defined as 1 unit (hereafter, abbreviated as xe2x80x9cUxe2x80x9d).
[Macromolecularization Method and Its Use]
In the process of producing phenolic compounds or aromatic amine compounds having increased molecular weights according to the present invention, the concentration of phenolic compounds or aromatic amine compounds is 0.01 to 90%, preferably 1 to 80%. The reaction temperature is 0 to 150xc2x0 C., preferably 0 to 100xc2x0 C. Further, the reaction pH is 7.0 to 12, preferably 7.5 to 10. The activity concentration of the enzyme to be used is 1 to 10,000 U/liter, preferably 10 to 2,000 U/liter. It is desirable that the enzyme activity concentration be adjusted depending on the purpose. That is, when more speedy macromolecularization and gelation or solidification is contemplated to be achieved, the reaction will be carried out at higher activity concentrations. On the other hand, when the reaction is carried out at lower activity concentrations, milder macromolecularization reaction will proceed, giving rise to a more homogeneous macromolecule solution as a liquid substance, and when the reaction is continued further, mild gelation reaction will proceed throughout the reaction mixture. When a suitable degree of macromolecularization is reached, the reaction can be terminated by the addition of alkali or alkali salts, such as NaOH, NH3, Na2CO3, and CaCO3, addition of acids such as hydrochloric acid, sulfuric acid, and nitric acid, addition of known enzyme inhibitors, or heat treatment such as that at 100xc2x0 C. for 15 minutes.
The gelled phenolic compounds or aromatic amine compounds can optionally be molten again by heating at 50 to 230xc2x0 C. Such a heat melting property is a useful property when the compounds are used in applications such as a dispersant, an adhesive and a coating composition. Also, it is possible to obtain phenolic compounds or aromatic amine compounds having very high molecular weights as a solution by addition of hot water or the like after the heat melting to disperse or dissolve the compounds above.
In order to accelerate the thermal curing, it is also possible to add polyols and the like such as furfuryl alcohol, sugars and etc. In order to have a physiologically active substance contained in the macromolecular compound to obtain an immobilized physiologically active substance or substance with a controlled release of physiologically active substance, it is also possible to carry out the macromolecularization with an antimicrobial compound, antiviral compound, a biotic repellent compound, an insecticidal compound or a metal ion coexisting, or to add an antimicrobial compound, antiviral compound, a biotic repellent compound, an insecticidal compound or a metal ion after the macromolecularization. As the antimicrobial compound, antiviral compound, biotic repellent compound, insecticidal compound or metal ion used for this purpose, there may be used many substances that have been known heretofore.
The macromolecularization reaction according to the present invention uses as an oxidation catalyst an enzyme having a polyphenol oxidizing activity and the oxygen in the air as an oxidizer, which makes it possible to apply the present invention to a wide field of applications. Further, when production of a large amount of macromolecule is contemplated, operations such as mechanical stirring of the reaction mixture and addition of air or oxygen to the reaction system are effective. Also, it is possible to carry out the reaction of the present invention in which oxygen is used as an oxidizer and the reaction in which hydrogen peroxide is used as an oxidizer simultaneously by adding to the reaction mixture peroxidase and hydrogen peroxide, or instead of hydrogen peroxide, an oxidase which can generate hydrogen peroxide and a substrate thereto.
[Phenolic Compounds or Aromatic Amine Compounds]
As the phenolic compounds or aromatic amine compounds to be macromolecularized in the present invention, there may be used any compound as far as the enzyme used in the present invention can oxidize it.
Specific examples of such phenolic compounds or aromatic amine compounds include lignin, lignosulfonic acid, humic acid, nitrohumic acid, tannin, catechin, gallic acid, urushiol, hesperidin, chlorogenic acid, hinokitiol, pyrocatechol, hydroquinone, t-butylhydroquinone, phenylhydroquinone, trimethylhydroquinone, ethyl 3,4-dihydroxycinnamic acid, pyrogallol, lauryl gallate, octyl gallate, syringic acid, ferulic acid, vanillin, o-vanillin, vanillic acid, vanillyl alcohol, ascorbic acid, 1,2-dihydroxynaphthalene, 2,3-dihydroxynaphthalene, 6,7-dihydroxy-2-naphthalenesulfonic acid, anthrarobin, alizarin, quinizarin, o-phenylenediamine, p-phenylenediamine, 3,4-diaminobenzophenone, o-anisidine, p-anisidine, o-aminophenol, p-aminophenol, 1,2-diaminoanthraquinone, and 1,4-diaminoanthraquinone.
Compounds other than these may also be used as a raw material for macromolecules or as a catalyst for the macromolecularization reaction as far as such compounds are substances that the enzyme used in the present invention can oxidize. Examples of such compounds include ABTS (2,2xe2x80x2-azobis(3-ethylbenzothiazoline-6-sulfonic acid)), bilirubin, isoascorbic acid, quercetin, rutin, guaiacol, 4-methoxyphenol, biphenol, 4,4xe2x80x2-ethylene-dianiline, methylhydroquinone, 1-hydroxybenzotriazole, 6-hydroxy-2,4,5-triaminopyrimidine, 4,5,6-triaminopyrimidine, 2,3-dihydroxypyridazine, 3,6-dihydroxypyridazine, 2,3-dihydroxypyridine, 4-hydroxy-3-methoxybenzoic acid, methyl 4-hydroxy-3-methoxybenzoate, 4,5-diamino-6-hydroxy-2-mercaptopyrimidine, 2,3-diaminopyridine, 2,5-dihydroxy-1,4-benzoquinone, 2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4-dihydroxy-3-cyclobuten-1,2-dione, 3-(3,4-dihydroxyphenyl)-L-alanine, 2-amino-3-hydroxypyridine, 3-amino-2-methoxydibenzofurane, 2,4-dimethoxyaniline, 2,5-dimethoxyaniline, 3,4-dimethoxyaniline, 2xe2x80x2,5xe2x80x2-dimethoxyacetophenone, 3xe2x80x2,4xe2x80x2-dimethoxyacetophenone, 1,4-dimethoxybenzene, veratrol, 2,3-dimethoxybenzoic acid, 2,5-diemethoxybenzoic acid, veratric acid, 3,4-dimethoxybenzyl alcohol, 3,4-dimethoxyphenethylamine, (3,4-dimethoxy-phenyl)acetic acid, (3,4-dimethoxyphenyl)acetonitrile, 4-allyl-2-methoxyphenol, 2-methoxy-4-propenylphenol, 2-methoxy-5-methylaniline, 2-methoxy-5-nitroaniline, 4-methoxy-2-nitroaniline, 3-methoxysalicylic acid, 3-methylcatechol, 4-methylcatechol, methylgallate, propylgallate, 3,4,5-trimethoxyaniline, 3,4,5-trimethoxyphenol, tropolone, purpurogallin, salicylaldoxime, 3-amino-5,6,7,8-tetrahydro-2-naphthol, 1,5-dihydroxynaphthalene, 3,5-dihydroxy-2-naphthoic acid, 4-hydroxy-1-naphthalenesulfonic acid, purpurin, 2,3-dihydro-9,10-dihydroxy-1,4-anthracenedione, and various azo dyes.
Also, in order to control the physical properties of the macromolecules, it is possible to use a plurality of such phenolic compounds or aromatic amine compounds in combination.
Upon producing macromolecularized phenolic compounds or aromatic amine compounds according to the present invention, quinone compounds may coexist which can be macromolecularized in a similar reaction path. Examples of such quinone compounds include anthraquinone-2-sulfonic acid, anthraquinone-1,5-disulfonic acid, anthraquinone-2,6-disulfonic acid, anthraquinone-2-carboxylic acid, 1-aminoanthraquinone, 2-aminoanthraquinone, anthrarufine, aminonaphthoquinone, 1,8-dihydroxyanthraquinone, camphorquinone, dehydroascorbic acid, 2-hydroxy-1,4-naphthoquinone, isatin, 5-nitroisatin, and various anthraquinone dyes. Also, it is possible to carry out air oxidation and macromolecularization simultaneously with enzymatic reaction under the coexistence of autooxidated substances, e.g., unsaturated fatty acids such as oleic acid and rinolic acid or unsaturated alcohols such as oleyl alcohol, or unsaturated alkyls such as squalene.
Of the macromolecularized phenolic compounds or aromatic amine compounds produced according to the present invention, particularly macromolecules of natural substances or derivatives thereof, such as lignin, lignosulfonic acid, humic acid, nitrohumic acid, tannin, catechin, gallic acid, urushiol, hesperidin, and hinokitiol, are highly useful because they are highly safe to environment as well as to humans so that making the best of their characteristics as high-molecular-weight compounds, they can be utilized in various fields of application such as thickeners, stabilizers, coagulants, emulsifiers, dispersants, water retainers, antioxidants, adhesives, concrete admixtures, dyes, coating materials, petroleum recovering agents, soil conditioners, blow-applied seed bearing surface soil stabilizers, deodorants, smell eliminators, agricultural chemical spreaders, feeding stuff binders, bactericides, antimicrobial agents, viral infection inhibitors, bioadhesion preventives. biotic repellents, insecticides, poultices, ink bases and wood treating agents. It should be noted that in these applications, various additive components usually used in each field can be employed in combination.
Further, in these fields of application, it is possible to develop applications of higher functions of the high-molecular-weight compounds by macromolecularizing natural or non-natural phenolic compounds or aromatic amine compounds under milder reaction conditions in the production process of the present invention to control the physical properties such as viscosity, adhesion, water retention, water solubility, water resistance, resilience, and strength or physiological effects.
Also, in accordance with the present invention, a method of disposing waste water is possible in which the enzyme having a polyphenol oxidizing activity is allowed to act on waste water containing phenolic compounds or aromatic amine compounds in the alkaline pH region so that the phenolic compounds or aromatic amine compounds in the waste water can be macromolecularized and readily concentrated and the macromolecularized phenolic compounds or aromatic amine compounds can be separated and removed from the waste water. As the field of application and reaction substrate for which such an application is particularly useful, there can be cited lignin or lignin derivatives in the field of paper pulp and dyes such as azo dyes or anthraquinone dyes in the field of coloring and dying. By carrying out coagulation precipitation treatment by addition of a coagulant, activated carbon treatment, or filtration treatment after the macromolecularization reaction, the phenolic compounds or aromatic amine compounds in waste water can efficiently be concentrated so as to be separated and removed from the waste water.
Also, in accordance with the present invention, a deoxygenation method or production of a deoxygenating agent is also possible by allowing the enzyme having a polyphenol oxidizing activity to act on phenolic compounds or aromatic amine compounds in the alkaline pH region to have dissolved oxygen consumed. Such deoxygenation method and deoxygenating agent are very useful since many natural or non-natural phenolic compounds or aromatic amine compounds can be utilized therein and the concentration of dissolved oxygen can be decreased quickly.
Also, in accordance with the present invention, impregnation of the enzyme having a polyphenol oxidizing activity together with phenolic compounds or aromatic amine compounds into wood and macromolecularization, in the wood, of the phenolic compounds or aromatic amine compounds and in addition the polyphenol compounds such as lignin already contained in the wood enables improvement in workability in a drying step after the wood impregnation treatment, improvement in the strength of wood which was decreased due to lignin decomposition by wood boiling treatment or high temperature steam injection treatment, and improvement in the effect of preventing wood cracking upon drying or freezing, prevention of growth of microorganisms due to maintenance or improvement in anaerobic environment in wood.
Also, in accordance with the present invention, by allowing polyphenol oxidase and a dye or its precursor on which the polyphenol oxidase can act, to act on wood, it is possible to produce a coloring substance in the wood or to co-macromolecularize the coloring substance and polyphenol compounds such as lignin already contained in the wood so that firmer dying or coloring treatment of wood can be achieved. In the above-described wood dying or coloring treatment, many polyphenol oxidases are known to bleach lignin, which is a coloring substance contained in wood and, hence, the wood dying or coloring treatment according to the present invention is very useful since it allows enzymatic bleaching and dying or coloring treatment simultaneously, thus reducing the number of process steps and improving the color tone.
Further, in accordance with the present invention, by adding the enzyme having a polyphenol oxidizing activity together with phenolic compounds or aromatic amine compounds to concrete and macromolecularizing the phenolic compounds or aromatic amine compounds in the concrete, it is possible to improve slump loss and concrete strength and suppress rust formation of ferro-reinforcement due to a decrease in the concentration of oxygen in the concrete.
Hereafter, the present invention will be described more concretely by representative examples which, however, are merely exemplary and the present invention should not be construed as being limited thereto.
In the following examples, the molecular weight analysis for macromolecularized phenolic compounds or aromatic amine compounds was carried out by HPLC using 50 mM of potassium phosphate buffer (pH 7.0) or 0.1 mM of sodium sulfate aqueous solution as an eluant, Shodex RI (differential refractive index detector, manufactured by Showa Denko) as a detector, and Shodex PROTEIN KW-802.5 (tandem, manufactured by Showa Denko) or a combination of Shodex PROTEIN KW802.5 (manufactured by Showa Denko) with Shodex OHpak SB-804HQ (manufactured by Showa Denko) as a column.